Glucagon is a major counterregulatory hormone that attenuates the inhibition of liver glucogenesis by insulin. Glucagon receptors are found primarily in liver, although their presence has been documented in kidney and adipose tissue. Type II diabetics have elevated levels of plasma glucagon and increased rates of hepatic glucose production. In fact, the rate of hepatic glucose production positively correlates with fasting blood glucose levels in type II diabetes. Therefore, antagonists of glucagon have the potential to improve insulin responsiveness in the liver, decrease the rate of gluconeogenesis and lower the rate of hepatic glucose output resulting in a decrease in the levels of plasma glucose.
Glucagon action is mediated by a G protein-coupled receptor that stimulates cyclic AMP accumulation via activation of Gs. G-protein coupled receptors are characterized by the ability of agonists to promote the formation of a high affinity ternary complex between the agonist, the receptor and the G-protein. The .alpha. subunit of the G-protein contains a guanine nucleotide binding site that, in the high affinity ternary G protein-receptor-agonist! complex, is occupied by GDP. In the presence of physiological concentrations of GTP, the GDP molecule in the guanine nucleotide binding site of the G protein is displaced by a GTP molecule. The binding of GTP dissociates the .alpha. subunit of the G protein from its .beta. and .gamma. subunits and from the receptor, thereby activating the G-protein to stimulate downstream effectors (adenylyl cyclase in the case of the glucagon receptor) and propagating the intracellular signal. Thus, the ternary complex is transient in the presence of physiological concentrations of GTP. Because the affinity of the agonist for the receptor-G protein complex is higher than its affinity for the uncomplexed receptor, one consequence of the destabilization of the ternary complex is a reduction in the affinity of the receptor for the agonist. Thus, the affinity of agonists for G-protein coupled receptors is a function of the efficiency with which the receptor is coupled to the G-protein. In contrast, antagonists bind with the same affinity to the receptor in the presence or absence of G-protein coupling.
G protein-coupled receptors such as the glucagon receptor are predicted to have seven transmembrane domains linked by hydrophilic loops. Extensive modeling and mutagenesis experiments show that the binding domain for small molecules is within the transmembrane helical domains, although peptide agonist binding also involves the hydrophilic extracellular domains. However, the intracellular loop domains are involved in coupling of receptor to G-proteins. Deletion of either the amino terminal or carboxyl terminal sections of the third intracellular loop led to loss of functional coupling of the .beta.-adrenergic receptor to Gs. However, these altered receptors maintained high affinity for agonist. The amino acids of the intracellular portions of the Glucagon receptor are shown as follows: loop 1, positions 167-173; loop 2; positions 250-262, loop 3, positions 332-349; and the carboxyl end, positions 404-477; of SEQ ID NO:3. Subsequent amino acid replacements in the third intracellular loop confirmed the role of this region in G protein interaction.
A human glucagon receptor has been cloned and expressed (D. MacNeil et al., 1994, Bioch. Biophys. Res. Comm. 198:328-334). We have characterized a mutant of the human glucagon receptor in which residues 252 to 259 in the second intracellular loop are deleted. The mutant protein is designated hGlu.DELTA.252-259 binding protein. When hGlu.DELTA.252-259 binding protein is expressed in COS cells, glucagon does not stimulate cAMP accumulation, suggesting that the hGlu.DELTA.252-259 binding protein does not couple to Gs upon agonist binding. However, the hGlu.DELTA.252-259 binding protein has higher affinity for glucagon than the wild type receptor, suggesting that the mutation locks the hGlu.DELTA.252-259 binding protein into a conformation with high affinity for agonist. Thus, hGlu.DELTA.252-259 binding protein is a high affinity glucagon binding protein that does not function as a glucagon receptor (ie., it does not transduce a signal).